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. 2024 Mar 4;19(3):e0297369. doi: 10.1371/journal.pone.0297369

Impact of pesticides exposure on Archachatina marginata snails in four Cameroon monomodal rainforest sites

Annick Niquaise Enangue Njembele 1,*, Maeva Arielle Patience Seppo Njembele 2, Emmanuel Henoch Dicka kwambe 1, Alexis Hamdja Ngoniri 3, Sylvie Ntyam epse Ondo 4, Kingsley Agbor Etchu 4
Editor: Selim Adewale Alarape5
PMCID: PMC10911591  PMID: 38437232

Abstract

Cameroon monomodal rainforest zone has a strong agricultural activity and is therefore exposed to pesticides. Furthermore, the area possesses climatic factors that favor the growth of Achatinadea snails known as African giant snails, a delicacy for the local population. The present study aimed to evaluate pesticides contamination (less vs more exposed areas) through assessment of exposure and impact on Achatinadea snails. Achatinadea snails were collected within intensive agricultural areas (Njombe and Kribi rural) and in areas with less agricultural activity (Ebodje and Dibombari). Collection was performed at night between July and September 2020 using an adapted square kilometer method. Type, number, weight, and size of the collected snails were analyzed and compared using Welsh’s One-way Analysis of variance (ANOVA). After removing the soft part from the shell, the presence of pesticides was determined using mass spectrometry. Histological analysis of kidney and ovo-testis was performed using eosin-hematoxylin staining. Results showed that the main variety of snails collected are Archachatina marginata. In areas with less agricultural activity, snails are bigger than those from more agricultural areas heavily using pesticides. Furthermore, pesticides detection showed that glyphosate, but not metalaxyl, is present in animals coming from all the collection sites. Cypermethrin was found in all the samples except in those from Dibombari. Histology revealed that the structure of the kidney and ovo-testis of snails from more exposed areas is impaired. In conclusion, this study revealed that some pesticides are transferred to snail and impair the structure of important organs.

Introduction

Cameroon is a country situated in Central Africa. It shares a large border in the north and west with Nigeria. Cameroon economy is mostly based on agriculture. Due to the climate, the country is divided into five agro-ecological zones (Sudano-Sahelian, Guinean High Savannah, Western Highland Plateau, Bimodal Rainforest and Monomodal Rainforest). The Monomodal Rainforest zone is known to have a strong agricultural activity in the country. Several industrial plantations such as CDC (Cameroon Development Corporation) and SOCAPALM (Cameroon oil palm company), PHP (Plantation Haut Penja) are found in this area. The local population are in majority farmers and they cultivate several crops for their consumption and to supply the urban area. In order to increase their productivity, farmers have turn to the use of pesticides [1–4], which use has increased the agricultural production in the area [4]. However, the best practice or safe use of pesticides is not yet well known by the majority of farmers, which has led to environmental biohazard [1, 3, 5–10]. Consequently, the area is highly prone to pesticides contamination. Additionally, the area is humid and favor the growth of land snails. Land snails are also attracted to the type of culture that are cultivated in the area such as palm oil nuts, bananas, and papaya trees, tomatoes, leafy vegetables, and diverse tuber and root plants such as cassava and sweet potatoes [11]. Snails are feeding on the fruits and leaves found on the ground of the farms [11]. The common edible snail group in the area is Achatinadea group also known as African giant snail. Achatinadea is the largest snail variety living in the soil. They are nocturnal and hermaphrodite (possess both types of reproductive organs) [12–14]. Among this group are found 3 species in Cameroon: Achatina achatina, Achatina fulica and Archachatina marginata [11, 14]. Various studies have shown that Achatinadea snail meat possesses many nutritional benefits [15–18] and is very appreciated by the local population [19]. Several studies have shown that pesticides and heavy metals are transferred to snails (land and freshwater snails) [20–24]. The land snail species that was mostly used as biomarker to monitor pesticides and heavy metals is Helix aspersa [21, 25, 26]. Most of the studies carried out on African giant snails are on their farming and nutritional aspect [15, 16, 27–35]. There are only a few studies on their potential biohazard contamination to human [33, 36–39]. The present study aims to evaluate pesticide contamination and the impact on Achatinadae snails in Cameroon agricultural areas. Our hypothesis is that Achatinadae snails could be contaminated and impaired by pesticides since they are living on soils of pesticide-exposed farms.

Materials and methods

Description of the study area

The study was performed within two regions: the South within the Ocean division (kribi rural and Ebodje), and the Littoral within Moungo division (Dibombari and Njombe) (Fig 1). According to the agroecological division of Cameroon, the study sites belong to the humid forest zone with mono-modal rainfall (consisting of one rainy season followed by one dry season). The Moungo division is characterized by the presence of volcanoes such as Mounts Manengouba and Koupe. The combination of all of these factors offers favorable climatic conditions for agriculture and also for the reproduction and development of Achatinadea snails. Ocean division is the nearest division to the Atlantic Ocean and the Congo Basin, that gives also the area a microclimate suitable for agriculture. More, within these two divisions are found several agricultural industries such as CDC, PHP, SOCAPALM that uses for some pesticides airplanes sprayer (done twice a week, CDC and PHP) and manual for SOCAPALM. These areas have small population. According to the 2022 census of the Cameroon National Institute of Statistic, these areas comprise 8,890 inhabitants for Njombe, 55,401 inhabitants for Kribi, 17,141 inhabitants for Dibombari and 6,000 inhabitants for Campo where Ebodje is a village. Local population is mostly farmers that cultivate food to supply major cities Douala and Yaoundé.

Fig 1. Map of the study area: Cameroon is divided into five agro-ecological zone.

Fig 1

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The study was performed within the Zone IV (in light green). Coordinates of the four collection areas coordinates were taken using a GPS and localization placed in Cameroon map showed here with red dots.

Sample collection

Achatinadea samples were collected into two types of sites within the monomodal rainforest area: intensive agricultural areas (Njombe and Kribi rural) versus area with less agricultural activity (Ebodje and the wild forest of Dibombari). Collection was performed at night between July to September 2020 using an adaptation of a square kilometer method [40, 41]. Coordinates of the collection areas were also taken using a GPS Triton 200.

Analysis of the general structure of snails

Collected snails of each group were counted as well as their weight was taken using sensible scale. General aspect: shell condition and the soft part (head and foot) were also analyzed.

Histology

Four snails from each collected groups were randomly chosen for histological studies. Snails were firstly left without food for 48 h to let them to digest all the food in their body. Animals were euthanized into boiled water and directly put on the fixative solution formaldehyde 4%. Dissection was done according to the protocol of Lőw et al. [42]. The soft part was removed from their shell then, ovo-testis and kidney were excised out. Furthermore, graded dehydration of the excised organs was done using 70% to 100% ethyl alcohol in subsequent steps. Xylene was used as a clearing agent. The organs were embedded in paraffin, sections were cut into 2–5μm thickness using rotary microtome and were stained with hematoxylin and eosin. Pictures of the section were taken with OMAX 40X-2500X LED Digital Trinocular Microscope with USB digital Camera (Irvine, California, USA) and analyzed with ImageJ 153a software.

Extraction and clean up for Gaz Chromatography–Mass Spectrometry (GC-MS)

Ten grams of the mix soft part of four snails coming from each collected group were mixed with 10 ml of acetonitrile, 2 g of MgSO4 and 0.5 g of NaCl. The mixture was vortexed for 1 min, and shaken for 15 min. Then, the samples were centrifuged at 3000 rpm for 5 min. One ml of the supernatant was mixed with 100 mg of Primary Secondary Amine (PSA) and 400 mg of MgSO4, vortexed for 2 min, and centrifuged as described above. The solvent was filtered on 0.25 μm thickness filter disk and subjected for GC-MS determination [43].

Gaz Chromatography–Mass Spectrometry (GC-MS) determination

Agilent Technologies 7890 GC system-coupled with MS-5977A MSD, Japan was used at the Faculty of Agriculture, Damanhour University, Egypt. GC-MS instrument with electron impact (EI) ionization, autosampler (AS), and computerized instrument control/data collection was used. Injection volume (2 μl) as spitless mode at 250 °C was conducted. An analytical column (30 m, 0.25 mm id, and 0.25 μm thickness of 5% phenyl methyl polysiloxane) was used and Helium was used as a carrier gas at a rate 1ml/min. Temperature program was started at 100 °C and ramped to 280 °C at a rate 10 °C/min. Software program used to estimate the output data.

Analytical method validation

All laboratory glassware were soaked for 12 hours in acid solution, washed carefully, purged with distilled water and acetone before use. All used chemicals were analytical grad. The limits of detection (LODs) ranged from 0.005 to 1.0 ng/ml for the examined pesticides. Extraction and clean up procedures were evaluated through recovery experiment which ranged from 94.0 to 95.5%. Also, procedural blank with no pesticides was performed. The calibration standards were done with coefficient of regression (R) ≥0.995. To employ reproducibility, each sample was duplicated.

Statistics

Mean of the weight and size of the animals were compared together, as well as Comparison of Vacuoles quantity estimation within the Eosin-hematoxylin-stained kidney by using Welsh’s One-way Analysis of variance with the p value p≤0.05 (ANOVA) with GraphPad Prism 9 software.

Ethics statement

All experiments therein have been conducted in accordance with current scientific bioethics law of Cameroon. All organisms unexposed to toxicants (i.e. unused and untested were given as food). Njombe and Ebodje sampling site locations are owned by the Institute of Agricultural Research for Development. The sampling site in Kribi rural is owned by the company SOCAPALM and had approved to perform the sampling. Concerning the remaining sampling location, Dibombari is not privately-owned or protected in any way and snails sampling at these locations did not require any specific permit. The field studies did not involve endangered or protected species.

Results

Result of collection

Two types of snail species were collected from the four sampling sites: Archachatina marginata and Achatina fulica. Archachatina marginata was the main species found at all the sites (Table 1). Further analyses were done on Archachatina marginata.

Table 1. Table of snails collected.

Animal Strain Archachatina marginata Archachatina fulica
Collection site
Weakly agricultural site Dibombari 32 0
Ebodje 18 2
Strongly Agricultural site Njombe 38 5
Kribi rural 36 1
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Collection was performed in four sites (weakly vs strongly pesticides exposed). Two types of snails were found: Archachatina marginata and Achatina fulica.

Pesticide measurements on snail soft part

Pesticides were measured on the soft part of snails in order to evaluate if they can be transferred to the flesh of the snail. The analysis of the chromatogram (Fig 2) showed in Table 2 that glyphosate is present in animals from all collection sites. In contrast, metalaxyl was not detected in animals from any of the collection sites. For cypermethrin, it was found in Njombe, Ebodje and Kribi rural samples but absent from Dibombari (Table 2).

Fig 2. Chromatogram of the pesticides measured and analyzed in Archachatina marginata by GC-MS analysis.

Fig 2

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10 g of the homogenate of four snails soft part coming from each collected group were used for mass spectrometry analysis. Peaks are the recorded mass spectra of each pesticide with corresponding retention time analyzed by the software GC-MS instrument used. The presence of several peaks on this chromatogram is due to the various degradation products and/or isomer of each pesticide molecule.

Table 2. Table of Pesticides residues levels (μg/g tissue) in whole body homogenate of collected Archachatina marginata snails.

Pesticides Glyphosate Metalaxyl Cypermethrin
Location
Dibombari 1.01399±0.7 ND ND
Ebodje 1.14834±0.812 ND 2.516±1.677
Njombe 0.35497±0.251 ND 3.035±2.037
Kribi Rural 0.09899±0.07 ND 10.90217±7.709
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10 g of the homogenate of four snails soft part coming from each collected group were used for mass spectrometry analysis. Value showed in the table are the mean of measured values coupled with their Standard Error of the Mean (SEM) obtained subsequently the analysis of pesticides peaks by the software of the GC-MS instrument. ND: not detected.

Effect of pesticide exposure on the general physiology of animals

Foot and shell, weight and length were examined to assess if the exposure to the different pesticides could affect the general physiology of Archachatina marginata snails. Results showed that snails collected in the less-exposed areas are heavier than those coming from more pesticide exposed areas: Dibombari 148 g and Ebodje 144 g versus Kribi rural 74 g and Njombe 35 g (Fig 3). It was also observed that the less exposed snails are taller than those more exposed: Dibombari 11 cm and Ebodje 10 cm versus Njombe 6 cm and Kribi rural 8 cm (Figs 4 and 5).

Fig 3. Pesticide exposure interfere with the weight of snails Archachatina marginata.

Fig 3

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Weight of collected Archachatina marginata snails were measured, compared together using Welsh’s One-way ANOVA and presented as histogram. Star indicates a statistically significant difference (* p≤0.05).

Fig 4. Pesticide exposure interfere with the size of snails Archachatina marginata.

Fig 4

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Size of collected Archachatina marginata snails were measured, compared together using Welsh’s One-way ANOVA and presented as histogram. Star indicates a statistically significant difference (* p≤0.05).

Fig 5. Snails coming from Dibombari are bigger than those from other sites.

Fig 5

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Picture of the different sizes of Archachatina marginata snails collected: Diverse sizes of Archachatina marginata snails (from the bigger to the smaller snail) coming from each site was put beside each other to have a visual comparison of histograms results.

Effect of pesticide exposure on ovo-testis structure of snails

Histological sections of the hermaphrodite reproductive gonad (ovo-testis) of Archachatina marginata snail was analyzed to determine whether pesticide exposure could disrupt their structures. Result of eosin-hematoxylin-stained sections showed a distinct disruption of the interstitial tissue on snail coming from Kribi rural and Njombe areas. The disruption is more noticeable with sections of snails collected from Njombe (Fig 6).

Fig 6. Pesticides exposure disrupts Archachatina marginata ovo-testis.

Fig 6

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Microphotograph of the Ovo-testis. Ovo-testis was excised out from the animals, sectioned and stained with eosin-hematoxylin. The structures of the conjunctive tissues were compared together. Njombe has the conjunctive tissue disrupted than the others. Blue arrow is showing the conjunctive tissue disruption. IT: Interstitial tissues, T: Tubules, TL: Tube Lumen.

Pesticide exposure on the structure of the kidney of snails

Histological sections of the kidney of Archachatina marginata snails were analyzed to determine whether pesticide exposure can impair their structures. Results of eosin- hematoxylin-stained sections of kidney showed a noticeable disruption of the structure of kidney lamina propria in samples from Njombe and Kribi rural areas (Fig 7). When quantity of vacuoles was compared together, a statistically significative increase in vacuoles were observed between samples coming from Njombe and Kribi rural areas when compared to Dibombari samples but not with Ebodje (Fig 8).

Fig 7. Pesticides exposure disrupts Archachatina marginata kidney.

Fig 7

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Microphotograph of the kidney. Kidney was excised out from the animals, sectioned and stained with eosin-hematoxylin. The structures of the lamina propria tissue and the presence of vacuoles were compared together. Njombe and Kribi rural have the conjunctive tissue disrupted than the others. As well the presence of a lot of vacuoles is visible in sections of the above-mentioned sites. The plain Arrow is showing the area with multiple vacuoles and the dash arrow is showing where Lamina propria is more disrupted. V: Vacuoles, LP: Lamina propria, TE: transitional epithelium.

Fig 8. Pesticides exposure increase vacuole quantity within kidney.

Fig 8

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Kidney was excised out from the animals, sectioned and stained with eosin-hematoxylin. Quantity of vacuole were counted, then compared together using Welsh’s One-way ANOVA and presented as histogram. Star indicates a statistically significant difference (* p≤0.05).

Discussion

The aim of the present study was to evaluate pesticide contamination and its impact on Achatinadea snails.

Animal collection showed that the snail species Archachatina marginata and Achatina fulica are the two species found in the collection areas. This result is supported by the survey of Tsayo et al that made a census of the types of snails found in the department of Mfoundi, Cameroon Center Region which is a region whose climatic conditions are similar to the study area [13]. On the other hand, in their study a similar number of individuals from the different species of snails was found whereas in our present study we have collected more Archachatina marginata snails. There are studies that mentioned Achatina fulica as invasive species [44–46]. We therefore expected to have individuals from this species in our collection, especially since they are the least consumed species in Cameroon, but we harvested less comparing to Archachatina marginata.

One of our hypotheses was that Cameroon most used pesticides are transferred into the flesh of snails. Previous studies with land snails including Achatinadea snails revealed the presence of heavy metals [20, 25, 37, 39] and pesticides [21, 24, 36, 47–49] on their flesh. Our present study also found that exposure to pesticides leads to their transfer into the flesh of Archachatina marginata snails. Indeed, the mass spectrometry results showed that glyphosate is present in the flesh of all the animals collected, regardless of the degree of agricultural activity. Indeed, glyphosate is an herbicide commonly used in most industrial or family-type fields to eliminate weeds in Cameroon. In contrast, metalaxyl was not detected in the sample of animals from any of the collection sites. Can probably be explained by the fact that metalaxyl is a fungicide used to fight against brown rot in cocoa farming and the fields where we collected snails are oil palm (Kribi rural) and plantain (Njombe) fields, not cocoa farms. Animals in Kribi rural and Njombe are not likely to be exposed to the metalaxyl. Moreover, since the recent prohibition of metalaxyl fungicide in Cameroon, several farmers tend more and more to abandon its use. Regarding cypermethrin, it was found in Njombe, Ebodje and Kribi rural samples but not in samples from Dibombari (Table 2). Besides, this study was focused on the presence/absence of pesticides in the Archachatina marginata snail which could be an indication of the transfer of these pesticides in the flesh of the animals collected. More work is needed to determine if consumption of contaminated snails could be a hazard for human health.

We also evaluated whether there is any impact of pesticides on Archachatina marginata snail physiology. We found that snails from pesticide heavily exposed areas are smaller in size and weight. This result suggests that the low weight and size could be a consequence of pesticide exposure. Indeed, the study of Wandan et al showed that exposure to high doses of the pesticide endosulfan do slightly negatively affect the weight of Achatina achatina snails which also belong to the Achatinadea group [50]. Other studies on different land snails have shown a correlation between snail growth and exposure to certain heavy metals like copper and lead [49, 51, 52]. Another hypothesis that can explain the low weight and size of snails from the high pesticide areas is that these areas also have more human activity, and the collection of snails for consumption is also higher. The small size of the snails could simply be due to the fact that they do not have enough time to grow and reach sizes seen in the less exposed areas of Ebodje and Dibombari that are wild forest areas. Assessing the impact of these pesticides on animal growth and the levels of growth hormone, in controlled laboratory conditions is warranted.

Finally, histological analysis of two organs, the kidney and the ovo-testis, showed a significant disruption in their structures. The disruption is more pronounced on the sections of the animals collected at Njombe which is heavily exposed to pesticides. Our results are in agreement with several studies carried out on animals exposed to heavy metals [38, 39] and certain pesticides such as glyphosate and chlorate pesticide [47, 53]. It would be interesting to evaluate the impact of these pesticides on the function of these two organs by measuring reproductive hormones and kidney enzymes.

Conclusion

The increase in the use of pesticides is a real problem in Africa, especially since their use is not well regulated. In order to find solutions to limit this pollution, our study was able to highlight that Archachatina marginata snails can be biological indicators to monitor soil pesticide pollution. Indeed, we confirmed the transfer of some pesticides in the flesh of snails caught in the usual collection areas and used to supply the market vendor. In addition, the results of this study showed that since there are pesticides in the meat of these animals, it represents a potential means of human contamination throughout the consumption of contaminated meat. The originality of our study lies in the use of a model that is very popular in Cameroon in real condition meaning: direct collection in usual areas by focusing on pesticides less vs highly contaminated sites. In contrary to other studies on Archachatina marginata that were performed in controlled areas. Furthermore, our result indirectly supports Archachatina marginata breeding in controlled area. Which is favorable for the specie conservation in the wild.

Supporting information

S1 File. Row data of morphology analysis.

Weight and size of Archachatina marginata snail were taken and then statistically analyzed using Welsh’s One-way Analysis of variance with the p value p≤0.05 (ANOVA).

(XLSX)

pone.0297369.s001.xlsx (24.2KB, xlsx)
S2 File. Row data of GPS values measured at the collection sites.

Coordinates of the four collection areas were taken by a GPS at night between July to September 2020 using an adaptation of a square kilometer method.

(XLSX)

pone.0297369.s002.xlsx (11.8KB, xlsx)
S3 File. Row data of mass spectrometry analysis.

Value showed in the table are the mean of measured values coupled with their Standard Error of the Mean (SEM) obtained subsequently the analysis of pesticides peaks coming from the homogenate of collected snails’ soft part by the software of the GC-MS instrument.

(XLSX)

pone.0297369.s003.xlsx (10.7KB, xlsx)
S4 File. Row data of histological analysis.

Microphotographs of the Ovo-testis and Kidney that were excised out from the collected Archachatina marginata snails, sectioned and stained with eosin-hematoxylin.

(PDF)

pone.0297369.s004.pdf (26.2MB, pdf)

Acknowledgments

We would like to thank Dr Rania Gabr, Horticulture Research Institute- Agriculture Research Center, Giza, Egypt, Dr Khaled Yassen, Faculty of Agriculture, Damanhour University, Egypt for the mass chromatography analysis. We acknowledge Edjengele Adolph, IRAD Ekona and Nangueu Belise Gladys, IRAD Kribi for their help in the interpretation of the mass Spectrometry result. We are also grateful to Rodrigue Fifen, Laboratory of Animal Biology, University of Yaoundé I for his help in the Histology analysis. Finally, would also like to acknowledge all the snails’ collectors at the various sites and particularly Pierre Njembele.

Data Availability

All relevant data are within the paper and Supporting information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Selim Adewale Alarape

22 Sep 2023

PONE-D-23-28780Impact of pesticides exposure on Archachatina marginata snails in four Cameroon monomodal rainforest sitesPLOS ONE

Dear Dr. enangue njembele,

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

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**********

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Reviewer #2: No

**********

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**********

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Reviewer #1: Study area: I recommend adding a description of samplingsites – for example - the main sources of pollution, industry, flow rates, population and others.

Results: The qualitative nature of the histological analysis. The histological analysis might have been more valuable to the reader if some index of severity were indicated. Currently, the scale is simply “positive” or “negative”. Some measure of severity would have helped.

The figure statements are not labelled properly and do not provide enough detail. I should not have to look to the body of the results to figure out what these letters indicate. Figures and figure statements should stand alone.

Reviewer #2: all my comments in an attached file

please supply the journal with the GM data (row data)

need to work on statistic and histology

there are questions in the attached file , please answer them

add figure on the peak of the GM for the pesticides with the retention time

**********

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Reviewer #2: No

**********

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PLoS One. 2024 Mar 4;19(3):e0297369. doi: 10.1371/journal.pone.0297369.r002

Author response to Decision Letter 0

11 Dec 2023

Enangue Njembele Annick Niquaise

IRAD/ Station specialized in Marine Ecosystem

PoBox 219 Kribi Cameroon

enangueannick@gmail.com

Kribi, 4th November 2023

Object: Resubmission of manuscript

reference PONE-D-23-28780

To the Editor of the journal PLOS ONE

Dear Editor,

We are honored to hear that our manuscript entitled:” Impact of pesticides exposure on Archachatina marginata snails in four Cameroon monomodal rainforest sites” has a chance to be published in your prestigious journal. We are grateful for your and the reviewers’ comments and positive evaluation on our manuscript. Here is the response letter that address point by point all the comments and questions raised.

Please find enclosed with this letter, the revised version of manuscripts (the highlighted and the unmarked versions), the revised version of figures and tables files. We also attached the row data file.

The changes made are considerably improved the manuscript and do hope that the revised version is now suitable for publication. We look forward to hearing from you.

Your sincerely,

Annick Enangue Njembele, PhD

 

Response to the editor and reviewers

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/fileid=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Answer: Changes were made accordingly

2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "These data were Presented in part at the 54th annual meeting of the Society for the Study of Reproduction, 15–18 December 2021, St. Louis, MO, USA". Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

Answer: The portion of the manuscript was presented as an oral poster. There was not a peer reviewed process to present at that conference. However, the abstract appeared in the program of the conference. Also, the poster was uploaded on the website of the conference since it was a remote presentation done during the COVID period. More, the mass spectrometry result was not available at the time of the presentation.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Answer: Minimal data will be uploaded while submitting the reviewed manuscript.

4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Answer: Figure 1 was modified Accordingly. There is no more need of copywriting permission. Actually, we now collaborated with a specialist in cartography that designed and provided a new version of the map. We also included that specialist as an author of this publication.

5. Please note that tables (should remain/ be uploaded) as separate "table" files"

Answer: Modifications were made accordingly

Reviewers' comments to the Author and Answers

Reviewer #1:

Study area: I recommend adding a description of sampling sites – for example - the main sources of pollution, industry, flow rates, population and others.

Answer: Modifications were made accordingly

Results: The qualitative nature of the histological analysis. The histological analysis might have been more valuable to the reader if some indexes of severity were indicated. Currently, the scale is simply “positive” or “negative”. Some measure of severity would have helped.

Answer: Results are more explained in the reviewed manuscript by using the appropriate words of parts of the tissues that we were able to identify. The name was added in the pictures. For the kidney, we counted the number of vacuoles and did the comparison between every location via Welsh’s one-way ANOVA in order to point out the difference / index of severity between each collection sites.

The figure statements are not labelled properly and do not provide enough detail. I should not have to look to the body of the results to figure out what these letters indicate. Figures and figure statements should stand alone.

Answer: Modifications were accordingly

Reviewer #2:

1-Please supply the journal with the GM data (row data)

Answer: Row data were attached with the reviewed version of the manuscript

2-Need to work on statistic and histology

Answer:

Statistic: We compared the values of size and weight of animals collected from the different locations by using welsh’s one-way ANOVA. That statistic technique was used since the size of the samples were different from each location. To strengthen the result obtained, we compared two by two the values (size and weight) of locations with Student t-test. More, to have an overview of what is shown in the histograms of weight and size of snail, the picture of Fig 5 is showing from the bigger to the smaller snails of each collection.

Histology: For the quality of the picture, we uploaded corrected versions of histology sections microphotograph by using PACE as recommended by the journal. Results were as well more explained in the reviewed manuscript by using the appropriate words of parts of the tissues that we were able to identify. Names was added in the pictures. For the kidney, we counted the number of vacuoles and did the comparison between every location via Welsh’s one-way ANOVA in order to point out the difference between each collection sites. We also added a scale to better appreciate the sizes of animals.

3- Add figure on the peak of the GM for the pesticides with the retention time

Answer: While sending back the result of Mass spectrometry analysis, I was given 3 files that comprised concentrations of analyzed pesticides in the flesh, the method and the chromatogram showed in Fig 2. They did not provide the chromatogram with the name and the retention time of pesticides. When asked recently, the laboratory responded they do not have anymore that information since it was done in 2022. I even seek help to some chemists that were no able to identify each pesticides analyzed since the original file of the chromatogram was missing. Is it possible to consider only the concentrations showed in table 2?

4- The point is not new

Answer: Actually, this is the first paper showing the presence of pesticides in African giant snails coming from the wild specifically from agricultural areas and where snails are often collected for consumption. This result is showing that snails sold in the market in Cameroon might be pesticides-contaminated, since collection is the major way to supply them in the market. It is interesting since that could be a potential biohazard for human. Moreover, the result of this paper will have an impact in local population since it showed the pesticide contamination in natural condition. Consequently, that will increase the awareness on those community to not perform wild snails’ collection but in contrary, increase their rearing. More, we showed a disruption on two pesticides sensible organs (kidney and ovotestis). This result could imply that pesticides might affect their reproduction and detoxification. There are two other papers that showed the presence of pesticides and their impact but that was after exposure in controlled environment the impact was not observed on kidney which is a new information that we showed.

5-How they did they send the sample to Egypt?

Answer: Snails samples were dried for better conservation before sending by plane.

6-The sequence and the plan of the paper are not clear

Answer: The goal of this study was to evaluate the impact pesticides exposure of snail collected in the field on their general morphology, on two pesticides sensible organs (kidney and ovo-testis) and to the determine their pesticides presence on the flesh of these animal. The sequence used in the manuscript was structured to answer the following questions:

� First, are there any changes on snails’ general morphology due to pesticides exposure in the wild?

� Second, can we see disruption within some pesticides-sensible organs?

� Third, if there is any disruption, pesticides can them be found in animal flesh?

We modified the manuscript according to the above structure/sequence.

7- The distribution of the histology is not enough and did not show any structure or the bar

Answer: Actually, the quality of the staining can allow us to see properly the structure of analyzed organs. Concerning the scale bar, we uploaded the PACE recommended version that can allow to see the bar. Concerning the distribution, I showed contaminated in the same line vs non contaminated.

8- I supposed to reject it but I gave them another chance

Answer: We are grateful for the second chance to our paper. We made as best as possible all the changes requested.

Attachment

Submitted filename: Response to Reviewers .docx

pone.0297369.s005.docx (26.1KB, docx)

Decision Letter 1

Selim Adewale Alarape

4 Jan 2024

Impact of pesticides exposure on Archachatina marginata snails in four Cameroon monomodal rainforest sites

PONE-D-23-28780R1

Dear Dr. ANNICK NJEMBELE,

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Acceptance letter

Selim Adewale Alarape

21 Feb 2024

PONE-D-23-28780R1

PLOS ONE

Dear Dr. Enangue Njembele,

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. Row data of morphology analysis.

    Weight and size of Archachatina marginata snail were taken and then statistically analyzed using Welsh’s One-way Analysis of variance with the p value p≤0.05 (ANOVA).

    (XLSX)

    pone.0297369.s001.xlsx (24.2KB, xlsx)
    S2 File. Row data of GPS values measured at the collection sites.

    Coordinates of the four collection areas were taken by a GPS at night between July to September 2020 using an adaptation of a square kilometer method.

    (XLSX)

    pone.0297369.s002.xlsx (11.8KB, xlsx)
    S3 File. Row data of mass spectrometry analysis.

    Value showed in the table are the mean of measured values coupled with their Standard Error of the Mean (SEM) obtained subsequently the analysis of pesticides peaks coming from the homogenate of collected snails’ soft part by the software of the GC-MS instrument.

    (XLSX)

    pone.0297369.s003.xlsx (10.7KB, xlsx)
    S4 File. Row data of histological analysis.

    Microphotographs of the Ovo-testis and Kidney that were excised out from the collected Archachatina marginata snails, sectioned and stained with eosin-hematoxylin.

    (PDF)

    pone.0297369.s004.pdf (26.2MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers .docx

    pone.0297369.s005.docx (26.1KB, docx)

    Data Availability Statement

    All relevant data are within the paper and Supporting information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

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